Prevalence of Peste des Petits Ruminant (PPR) virus in small ruminant of Gujarat and its characterization by RT-PCR / RFLP and SSCP profiles.
Tiwari, Ashish (2004) Prevalence of Peste des Petits Ruminant (PPR) virus in small ruminant of Gujarat and its characterization by RT-PCR / RFLP and SSCP profiles. Masters thesis, College of Veterinary Science and Animal Husbandry, Anand Agricultural University, Anand.
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Peste des petits ruminants (PPR) is an acute viral disease of small ruminants caused by a Morbillivirus and characterized by fever, oculonasal discharges, stomatitis, diarrhoea and pneumonia. In India, PPR was first reported in 1987 from Tamil Nadu and for several years, the disease was thought to be restricted to southern India only, however in 1994, and after a series of PPR outbreaks was reported from many northern states as well as from West Bengal. The disease causes severe losses to small ruminant production and is presently considered as one of the major threats to about 200 million small ruminant population of the country. The present study was planned to carry out a seroprevalence study of Peste des petits ruminants virus (PPRV) in Gujarat state and to derive estimates of overall, zonewise, districtwise, yearwise and species wise seroprevalence. The study was also aimed to detect PPRV in clinical samples using sandwich-ELISA and to derive estimates of overall, location wise, species wise and age wise prevalence of PPRV. Also, RT-PCR was standardized and applied for detection of PPRV from field samples and its sensitivity and specificity was compared with sandwich-ELISA. Further, an attempt was made to assess possible genetic variation among the field and vaccine PPRVs by RT-PCR /RFLP and SSCP profiles. A total of 520 Serum samples collected from 17 districts of the five different geographical zones viz. North, South, Central, Saurashtra and Kachchh of Gujarat state were screened for PPR specific antibodies using PPR c-ELISA kit developed by National Rinderpest Laboratory, Division of Virology, IVRI, Mukteswar. One hundred ninty one samples were positive for PPR antibodies yielding an overall seroprevalence of 36.70 per cent in the small ruminant population included in the study. Among the zones, the seroprevalence was 53.67, 33.33, 19.00, 43.18 and 15.00 per cent in North Gujarat, South Gujarat, Central Gujarat, Saurashtra and Kachchh zones, respectively. Among the districts sampled, highest seroprevalence of PPR was observed in goat population of Bharuch district (100%) and the lowest seroprevalence was recorded in goat populations of Panchmahal, Navasari and Surat districts (0.00%, each). The seroprevalence in other districts ranged from 15.00 to 68.18 per cent, of which Bhavnagar (68.18%), Mehsana and Sabarkantha (67.50%, each) showed higher seroprevalence rates. For the years 2003 and 2004, seroprevalence of PPRVin small ruminants was 34.04 and 38.25 per cent, respectively. In the year 2003, seroprevalence of PPRV in sheep and goat was 22.78 and 42.20 per cent, respectively. These values for the year 2004 were 54.02 and 32.65 percent, respectively. Specieswise seroprevalence of PPRV in sheep and goat was 39.16 and 35.59 per cent, respectively. A total of 30 clinical samples from 25 small ruminants (10 sheep and 15 goats) suspected of PPRV, were tested for PPRV antigen using PPR sandwich-ELISA kit developed by National Rinderpest Laboratory, Division of Virology, IVRI, Mukteswar. Fifteen animals were found positive yielding an overall incidence rate of 60.00 per cent. Out of 20 animals (including 10 sheep and 10 goat) suspected of PPR at Sheep Breeding Farm, Patan, 11 (55.00%) were found positive; whereas, four (80.00%) out of five goats suspected of PPR at Veterinary Polyclinic, Vadodara, yielded positive results. Incidence rate of PPR by sandwichELISA was 30.00 and 80.00 per cent in sheep and goats, respectively. Out of the 25 animals, five, eight, nine and three animals belonged to age groups of 0-24, 24-48, 48-72, and >72 months, respectively, showing the respective incidence of PPRV as 100, 62.50, 44.44 and 33.33 per cent. All the 30 clinical samples, a reference vaccine virus (Sungri isolate) and a blood sample from apparently healthy goat were processed for RNA extraction using TRI Reagent®. RNA samples showing acceptable purity and concentration were reverse transcribed with random hexamers to generate cDNA templates, which were subjected to PCR amplification using the F-gene specific primer pairs F1-F2 and F1b-F2d. Reference vaccine virus as well as eight (26.67%) of the 30 clinical samples, including four each of blood and swab samples produced approximately 372 bp and 448 bp expected amplicons with primer pairs F1-F2 and F1b-F2d, respectively. The amplicons from all the positive samples migrated similarly in the gel at respective locations expected for the two primer pairs used. Twenty two (73.33%) samples including 21 blood samples and one oral swab suspension as well as the blood sample taken from apparently healthy goat failed to produce the targeted amplification with both the primer pairs. Out of 30 clinical samples tested for PPRV, 15 and eight samples were positive by sandwich-ELISA and RT-PCR, respectively. None of the samples negative in sandwich- ELISA yielded positive amplification in RT-PCR. Relative to sandwich-ELISA, sensitivity and specificity of the RT-PCR was 53.33 and 100.00 per cent, respectively. Overall agreement between the two tests was 76.67 per cent. RT-PCR products amplified with primer pair F1-F2, were further digested with three restriction enzymes viz. Msp-I, Pst-I and EcoRI for RFLP analysis. RT-PCR products of all the eight positive samples and the reference vaccine produced similar RFLP patterns with each of the three REs. Digestion of RT-PCR products with Msp-I yielded two fragments of approximately 116 bp and 256 bp each. Digestion with Pst-I resulted in two fragments of 155 bp and 217 bp each. None of the sample was digested with EcoRI, thus showing the original RT-PCR product. To detect any possible mutation in the targeted sequence of PPRV F-gene, RT-PCR products amplified with primer pair F1-F2, were subjected to SSCP analysis on 0.5X MDE™ gel (with 10% glycerol) in 0.5X TBE buffer system. Seven out of the eight RT-PCR positive field samples and the reference vaccine virus (Sungri isolate) yielded similar SSCP profiles producing three bands each, one corresponding to faster migrating non-denatured PCR product and two relatively slow migrating bands corresponding to single stranded DNA. One sample (P/2) collected from a goat, generated a SSCP profile different from all the other field samples as well as the vaccine virus. It yielded five bands, one of which corresponded to non denatured PCR product and the other four bands of single stranded DNA, possibly indicating a genetic variation in the targeted F-gene 372 bp amplicon.
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